J Pest Sci (2004) 77: 11–15 DOI 10.1007/s10340-003-0020-7
O R I GI N A L A R T IC L E
S. Stankovic´ Æ A. Zabel Æ M. Kostic Æ B. Manojlovic S. Rajkovic
Colorado potato beetle [Leptinotarsa decemlineata (Say)] resistance to organophosphates and carbamates in Serbia
Received: 22 February 2003 / Published online: 10 January 2004 Ó Springer-Verlag 2004
Abstract Leptinotarsa decemlineata (Say), the Colorado potato beetle (potato beetle), is the most destructive potato pest in Serbia. Up to four pesticide treatments are necessary for its control. Insecticide resistance of the potato beetle in Serbia is well-investigated and documented, especially to organophosphates and carbamates. Toxicity of chlorpyriphos (organophosphates) and carbosulfan (carbamates) was investigated by topical application, using adults of the first generation from nine field populations. Adults of all populations possessed a certain level of resistance to both insecticides, with very low regression slopes (0.50–1.09 for chlorpyriphos and 0.57–0.85 for carbosulfan). Resistance ratios for chlorpyriphos ranged from 1.0 to 109.7 and from 1.0 to 52.8 for carbosulfan. There was no correlation between resistance to chlorpyriphos and carbosulfan. The key modes of resistance to organophosphates and carbamates are metabolism [overproduction of aliesterase (ALiE), a carboxylesterase] and altering the target site of acetylcholine esterase (AChE). Biochemical research of potato beetle resistance to organophosphates and carbamates, based on activity of ALiE and AChE, proved that the key mode of resistance to organophosphates was modification of ALiE and AChE to carbamates. Our results correspond to this research concerning the lack of correlation between resistances to these two insecticides. A certain level of resistance of the potato beetle to organophosphates and carbamates is widespread in Serbia. Different modes of resistance suggest the possibilities of control of this pest, such as choice of insecticide and insecticide rotation, wherever possible. S. Stankovic´ (&) Institute for Science Application in Agriculture 29, Novembra 68B, Belgrade, Yugoslavia E-mail:
[email protected] A. Zabel Æ B. Manojlovic Æ S. Rajkovic Institute for Plant Protection and Environment, Belgrade, Yugoslavia M. Kostic Institute for Medicinal Plant Research, Belgrade, Yugoslavia
Keywords Colorado potato beetle Æ Resistance Æ Insecticide Æ Organophosphates Æ Carbamates
Introduction The Colorado potato beetle Leptinotarsa decemlineata (Say) (Insecta: Coleoptera: Chrysomelidae) is a limiting factor for the production of potatoes (Hare 1990). It causes potato defoliation (most intensive during blooming), which results in extremely high losses of potato yield (Nault and Kennedy 1998). Hare (1980) and Zehnder and Evanylo (1989) showed that significant yield decrease is a result of defoliation several weeks prior to harvest. Control of the potato beetle costs the worlds’ producers hundreds of millions of dollars every year (Hu et al. 1998). Pesticide control in most regions with high population densities of potato beetle still has a primary role in control of the potato beetle, since alternative measures are not efficient enough (Hare 1990). In Serbia, the potato beetle produces two generations a year, leading to a high density. In order to ensure a normal yield, potato in Serbia is treated with insecticides three to four times a year (Zabel and Kostic 1988; Zabel et al. 2000a, 2000b). The remarkable capability of the potato beetle to adapt to adverse environmental conditions, by developing resistance to insecticides, causes an increase in production costs, contamination of the natural environment and disturbs the balance in biocenosis (Hare 1990; Nault and Kennedy 1998). The insecticides carbosulfan and chlorpyriphos, from the group of carbamates and organophosphates, respectively, have been applied to control potato beetles for years. They act as inhibitors of acetylcholine esterase (AChE), building the enzyme-inhibitor complex. The complex building reaction is not irreversible, but the speed of reactivation is 105–106 times slower as compared to the reaction in case of acetylcholine (ACh) and AChE, so the enzyme is not capable of dissolving ACh efficiently. Organophosphates
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and carbamates, although having similar modes of action, also have significant differences as a result of different bindings in the enzyme-activation center (Corbett 1984). Resistance to organophosphates and carbamates has been well investigated. It has been determined that increased production of aliesterase (ALiE) is a significant mode of resistance, along with the change of AChE activity (Gunning and Moores 2001). In Serbia, Sˇestovic´ (1972) obtained the first quantitative data concerning resistance to organophosphates and carbamates. Further investigations on the resistance of the potato beetle followed (Peric´ et al. 1979; Sˇestovic´ and Peric´ 1977). Zabel (1991) determined a significant level of resistance of the third-larval instar to organophosphates and carbamates in a large number of localities in Serbia. Peric´ et al. (1997a, 1997b) determined resistance of several populations to carbamates and organophosphates, as did Zabel et al. (1997a, 1997b, 1998).
Materials and methods Test insects First-generation male and female adult potato beetles were used in this investigation. They were collected from the field and placed in cages (where food was given), then transferred to the laboratory and maintained at 5–8°C. After setting up the experiment, test insects were kept in microclimate chambers (Danfoss, EKH 20) in controlled conditions: t = 26±1°C, relative humidity = 60% and light regime=16/8 h(day/night). Food was again given during the experiment. [Test insects were collected from several localities, covering a large part of the country (Dobanovci, Majur, Malosˇ isˇ te, Recˇka, Orid, Pozˇarevac, Ruma, Toljevac and Vrsˇ ac). Due to the absence of a normally susceptible laboratory strain, populations from Orid and Toljevac were taken as reference populations. In the following year, four representative populations were investigated (Dobanovci, Majur, Recˇka, Toljevac).]
Insecticides The toxicity of insecticides chlorpyriphos and carbosulfan was investigated. Chlorpyriphos had a technical grade of 97.40% and carbosulfan a technical grade of 91.6% and Posse 25 EC (230 g/l am) (FMC, Philadelphia-Zupa, Krusevac). Technical grades were obtained from the Department of Phytopharmacy at the Institute for Plant Protection and Environment, Belgrade, where investigations on contents of technical grades and preparations were conducted.
Methods Toxicity for the potato beetle was determined by using a topical application method (FAO 1974). Using a microliter pipette ‘Socorex’, a 1-ll drop of an acetone solution of insecticide was applied to the ventral thorax sternite between coxes. Ten insects were treated and transferred into petri dishes. The accuracy of the pipette was 0.1 ll. Insects in checks were treated with acetone. Mortality was assessed daily. For calculations, the number of dead insects was included. Abbott’s formula was used for correction of results for mortality (Abbott 1925). Regression (ld-p) lines, values for LD50, with 95% fiducial limits, were determined using probit analysis (Finney 1971) and a computer program (Raymond 1985). Experiments were conducted in four replications, using at
least five concentrations of insecticides. Results were given as micrograms of active material per insect. Resistance ratios (RRs) were determined by comparing LD50 values of every population with LD50 values of reference populations. In addition to comparing the population values of LD50, it is also relevant to compare their intervals.
Results and discussion Susceptibility differences among adults from populations tested using chlorpyriphos are significant (Table 1), antedated from RR values(1.6–109.7). Most susceptible are populations from Orid, Pozˇarevac and Toljevac, whose LD50 intervals overlap. Populations from Ruma, Malosˇ isˇ te and Recˇka are more resistant and classified in the same group, since their LD50 intervals overlap. The most frequently rendered level of resistance is manifested in populations from Majur, Vrsˇ ac and Dobanovci where, except for the resemblance of LD50, the two populations have close interval values. The most sensitive population (Orid) and one closely susceptible population (Toljevac) have low and approximately similar slopes. Population Malosˇ isˇ te is characteristic for the lowest slope (0.50), while population Dobanovci is characteristic for a relatively high slope value. Population Dobanovci has two plateaus, where, by increasing the dose 20-fold, mortality rises four-fold. Two plateaus are also characteristic for the population Malosˇ isˇ te, when doses are increased five- and ten-fold. A significant plateau within the population Recˇka is a result of a ten-fold increased dose (Stankovic´ 2002). According to results obtained in the second year of investigation illustrated in Table 2, the difference between investigated populations is determined concerning this organophosphorous insecticide. Populations Majur, Recˇka and Dobanovci were 2.5–5.3 times more resistant as compared to the population Toljevac. A significant difference in LD50 intervals exists only between the populations Toljevac and Dobanovci. Regression slopes differ significantly and range from 0.53 to 1.63. The lowest slope is characteristic of the most susceptible population (Toljevac), and it rises as resistance of a population increases. The slope of the population Dobanovci is significantly higher than that
Table 1 Chlorpyriphos toxicity to Colorado potato beetle adults (1998) Population
Slope (b)
LD50 (lg/insect) (95% fiducial limits)
Resistance ratio (RR)
Dobanovci Majur Toljevac Recˇka Malosˇ isˇ te Pozˇarevac Ruma Orid Vrsˇ ac
1.09 0.93 0.66 0.76 0.50 0.98 0.84 0.62 0.75
7.68 2.10 0.15 0.46 0.68 0.11 0.53 0.07 4.39
109.71 30.00 2.14 6.57 9.71 1.57 7.57 1.00 62.71
(5.12–1206) (1.32–317) (0.06–0.30) (0.23–0.97) (0.21–1.64) (0.06–0.19) (0.30–0.95) (0.03–0.15) (2.27–10.36)
13 Table 2 Chlorpyriphos toxicity to Colorado potato beetle adults (1999)
Table 4 Carbosulfan toxicity to Colorado potato beetle adults (1999)
Population
Slope (b)
LD50 (lg/insect) (95% fiducial limits)
Resistance ratio (RR)
Population
Slope (b)
LD50 (lg/insect) (95% fiducial limits)
Resistance ratio (RR)
Dobanovci Majur Toljevac Recˇka
1.63 0.91 0.53 0.57
2.96 1.43 0.56 1.42
5.26 2.55 1.00 2.52
Dobanovci Majur Toljevac Recˇka
0.72 0.77 0.73 0.53
10.14 (5.38–24.97) 1.76 (1.00–3.54) 3.01 (1.68–5.85) 2.35 (0.82–5.62)
5.75 1.00 1.71 1.33
(2.09–3.99) (0.90–2.32) (0.24–1.32) (0.53–3.12)
Table 3 Carbosulfan toxicity to Colorado potato beetle adults (1998) Population
Slope (b)
LD50 (lg/insect) (95% fiducial limits)
Resistance ratio (RR)
Dobanovci Majur Toljevac Recˇka Malosˇ isˇ te Pozˇarevac Ruma Orid Vrsˇ ac
0.61 0.81 0.85 0.61 0.57 0.84 0.64 0.72 0.78
5.39 9.47 1.64 1.79 0.70 0.48 3.62 0.18 2.84
30.04 52.80 9.16 9.96 3.88 2.68 20.20 1.00 15.85
(2.41–16.03) (5.27–19.37) (0.99–2.81) (0.69–5.35) (0.33–1.42) (0.29–0.83) (1.77–9.37) (0.10–0.32) (1.56–5.93)
of the others. One plateau is present at population Majur (when the dose increases six-fold) and Recˇka (2.5-fold increased dose) (Stankovic´ 2002). In Serbia, organophosphoric compositions have been applied for a long time in control of the potato beetle, especially phosmet, fosalon and chlorpyriphos; thus, big differences in resistance can be expected, especially considering the history of insecticide application at certain localities (Dobanovci). The results obtained are similar to those determined for chlorpyriphos by Peric´ et al. (1997b), which are LD50 intervals of 1.7–16.8 lg/insect and intervals with a regression slope of 0.54–1.66. Differences in susceptibility to carbosulfan among investigated populations have also been determined (Table 3), with RR = 2.7–52.8. Populations Orid, Pozˇarevac and Malosˇ isˇ te also show the highest susceptibility to this insecticide. There is an exception concerning the susceptibility on other insecticides, including chlorpyriphos (Stankovic´ 2002): the population Toljevac, according to its LD50 and intervals, can be classified into a group of resistant populations. Also adhering to this group are the populations Recˇka, Vrsˇ ac, Dobanovci and Majur. The population Toljevac is characteristic of extremely distinct gradation, whereby a ten-fold increased dose leads to a 23% decrease of mortality. A somewhat slighter gradation, under the same dose increase, is present in population Malosˇ isˇ te (Stankovic´ 2002). Regression slopes for carbosulfan are generally lowest and do not exceed 0.85 (Toljevac). The lowest slope is, as in most previous surveys, determined in the population Malosˇ isˇ te (0.57). Investigated populations did not show significant differences in susceptibility to carbosulfan in 1999 (Table 4), although the population Dobanovci is
assigned the highest LD50 (10.14 lg/insect). Differences in LD50 intervals are not present. Population Toljevac this year showed significantly low susceptibility, while population Majur was significantly more susceptible. Regression slopes are equal and moderate (0.53–0.77). These values are in accordance with results obtained in the previous year. Majur is the only population characteristic of presence of bend when the dose is increased five-fold (Stankovic´ 2002). Resistance to carbamates has been determined in papers by numerous authors in Serbia: Indic´ et al. (1995,1997), Peric´ et al. (1979, 1997a, 1997b), Sˇestovic´ (1972), Sˇestovic´ and Peric´ (1977), Zabel (1991), Zabel et al. (1997a, 1997b), so that the determined resistance of most of the populations is not unexpected. Levels of LD50 obtained in 1998 and 1999 are significantly lower than results obtained more recently by Peric´ et al. (1997b) (LD50 in interval 17.8–211.3 lg/insect); therefore, it is possible to assume that the introduction of insecticides possessing different modes of action would decrease the total level of resistance. A certain level of resistance is selected, so populations have stabile and distinct resistance. Numerous investigators all over the world have determined the presence of resistance of potato beetle populations to carbamates and organophosphates. Harris and Svec (1976) determined the low toxicity effects of all organophosphates and carbaryl on potato beetle populations. Turnbull et al. (1988) determined high resistance to carbofuran of six populations, and moderate resistance to azinphosmetil of all investigated populations. Roush et al. (1990) determined significant levels of resistance to carbofuran and azinphosmetil, as did Bishop and Grafius (1991). Olson et al. (1996) determined susceptibility to azinphosmetil for 9 of 35 populations, while the others were significantly or moderately resistant. Stewart et al. (1997) determined significant resistance to carbosulfan and azinphosmetil. Argentine et al. (1994) and Miyo et al. (1999) determined that resistance to azinphosmetil is inherited by an autosomal gene, incompletely dominant. Heim et al. (1990) and Miyo et al. (1999) determined that mechanism of inheritance of resistance to carbosulfan is controlled autosomally, partially dominant. Basic mechanisms of resistance to these two groups of insecticides are hydrolytic detoxification and change of AChE susceptibility. The most significant hydrolytic enzymes are phosphoesterases and carboxylesterases (ALiE, a non-specific esterase) and represent basic factors
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of resistance to organophosphates. It is confirmed in the investigation of potato beetle populations’ resistance to quinalphos by Zabel (1991), who determined a positive correlation between results obtained by Biotest (done with fourth larval instar) and measures of ALiE activity (with a-naphtyl acetate). The role of esterase in potato beetle resistance was also confirmed by Anspaugh et al. (1995), Argentine et al. (1994), Olson et al. (2000) and Zhao et al. (2000). The impact of non-specific esterase on the level of resistance to carbamates was not confirmed (Miyata 1983), until proved by Zabel (1991) with the potato beetle. AChE as a mode of action of organophosphates and carbamates was the subject of changes that led to different levels of transformation. Modified forms of AChE present in different species differ among themselves. Several authors (Anthony et al. 1998; Barber et al. 1999; Hama 1983; Hassall 1990) indicated that modified AChE (MACE) causes resistance to carbamates and organophosphates. Investigation of resistance to organophosphates and carbamates showed that the activity of potato beetle AChE is very distinct and easily measurable. Total AChE activity of fourth larval instar correlated with determined resistance to carbaryl (Zabel 1991). Results from Toljevac concerning the individual level of resistance to chlorpiriphos and carbosulfan suggest that although modes of action are the same, the modes of resistance to these insecticides differ, which is not a rare case (Miyata et al. 1999). According to these and results obtained by Zabel (1991), it is possible to conclude that the basic mode of resistance of populations to carbamates in Serbia is a modification of AChE susceptibility, and in some part, modified hydrolytic detoxification. In addition to this conclusion is the fact that the population Toljevac is resistant to carbosulfan, and at the same time highly susceptible to imidacloprid (Stankovic´ 2002).
References Abbott WS (1925) A method for computing the effectiveness of an insecticide. J Econ Entomol 18:265–267 Anspaugh DD, Kennedy GG, Roe RM (1995) Purification and characterization of a resistance-associated esterase from the Colorado potato beetle, Leptinotarsa decemlineata (Say). Pestic Biochem Physiol 53:84–96 Anthony NM, Brown JK, Feyereisen R, ffrench-Constant RH (1998) Diagnosis and characterization of insecticide—insensitive acetylcholinesterase in tree populations of the sweet potato whitefly, Bemisia tabaci. Pestic Sci 52:39–46 Argentine JA, Zhu KY, Lee SH, Clark JM (1994) Biochemical mechanisms of azinphosmethyl resistance in isogenic strains of Colorado potato beetle. Pestic Biochem Physiol 48:63–78 Barber MD, Moores GD, Tatchell GM, Vice WE, Denholm I (1999) Insecticide resistance in the currant-lettuce aphid, Nasonovia ribisnigri (Hemiptera: Aphididae) in the UK. Bull Entomol Res 89:17–23 Bishop BA, Grafius E (1991) An on-farm insecticide resistance test kit for Colorado potato beetle (Coleoptera: Chrysomelidae). Am Potato J 68:53–64 Corbett A (1984) The biochemical mode of action of pesticides, 2nd edn. Academic, New York
FAO (1974) Recommended methods for the detection and measurement of resistance of agricultural pests to pesticides. Tentative method for adults of the Colorado potato beetle, Leptinotarsa decemlineata (Say). FAO method no. 12. FAO Plant Prot Bull 22:112–116 Finney DJ (1971) Probit analysis, 2nd edn. University Press, Cambridge Gunning R, Moores G (2001) Insensitive acetylcholinesterase as sites for resistance to organophosphates and carbamates in insects: insensitive acetylcholinesterase confers resistance in Lepidoptera. In: Ishaaya I (ed) Biochemical sites of insecticide action and resistance. Springer, Berlin Heidelberg New York, pp 221–239 Hama H (1983) Resistance to insecticides due to reduced sensitivity of acetylcholinesterase. In: Georghiou GP, Saito T (eds) Pest resistance to pesticides. Plenum, New York, pp 299–332 Hare DJ (1980) Contact toxicities of ten insecticides to Connecticut population of the Colorado potato beetle. J Econ Entomol 73:230–231 Hare DJ (1990) Ecology and management of the Colorado potato beetle. Annu Rev Entomol 35:81–100 Harris CR, Svec HJ (1976) Susceptibility of the Colorado potato beetle in Ontario to insecticides. J Econ Entomol 69:625–629 Hassall KA (1990) Biochemistry and uses of pesticides, 2nd edn. VCH, New York Heim DC, KennedyGG, Van Duyn JW (1990) Survey of insecticide resistance among North Carolina Colorado potato beetle (Coleoptera: Chrysomelidae) Populations. J Econ Entomol 83:1229–1235 Hu JS, Gelman DB, Bell RA, Loeb MJ (1998) In vitro rearing of Edovum puttleri, an egg parasitoid of the Colorado potato beetle—development from egg through the pupal stage. BioControl 43:1–16 Indic´ D, Klokocˇar- Sˇmit Z, Sˇestovic´ M, Peric´ I, Graovac M (1995) Toxicity of Insecticides to adults and larvae of Colorado potato beetle, Leptinotarsa decemlineata (Say). Pesticidi 10:213– 218 Indic´ D, Klokocˇar- Sˇmit Z, Orbovic´ B, Sˇestovic´ M, Peric´ I (1997) Ovicidal effect on insecticides used in control of Leptinotarsa decemlineata (Say). Acta Hortic 462:391–396 Miyata T (1983) Detection and monitoring methods for resistance in arthropods based on biochemical characteristics. In: Georghiou GP, Saito T (eds) Pest resistance to pesticides. Plenum, New York, pp 99–117 Miyata T, Kato Y, Nomura M (1999) Mechanism of negatively correlated cross-resistance in the green rice leafhopper, Nephottetix cincticeps (Uhler). 14th International Plant Protection Congress, Jerusalem, Israel (abstract) Miyo T, Keil CBO, Hough-Goldstein JA, Oguma Y (1999) Inheritance of resistance to esfenvalerate in Colorado potato beetle (Coleoptera: Chrysomelidae). J Econ Entomol 92:1031– 1038 Nault BA, Kennedy GG (1998) Limitations of using regression and mean separation analyses for describing the response of crop yield to defoliation: a case study of the Colorado potato beetle (Coleoptera: Chrysomelidae) on potato. J Econ Entomol 91:7– 20 Olson E, Dively G, Nelson J (1996) Survey of susceptibility to imidacloprid (Admire) in Colorado potato beetle (Coleoptera: Chrysomelidae). Resistant Pest Management Newsletter 8: http://www.msstate.edu/Entomology/v8n1/art14.html Olson E, Dively G, Nelson J (2000) Baseline susceptibility to Imidacloprid and Cross-resistance patterns in Colorado potato beetle (Coleoptera: Chrysomelidae) populations. J Econ Entomol 93:447–458 Peric´ I, Sˇestovic´ M, Milosˇ evski N (1979) Variranje osetljivosti krompirove zlatice prema insekticidima u jednom regionu. X jubilarno savetovanje o primeni pesticida-Porecˇ. Zbornik Radova 2:445–450 Peric´ I, Milosˇ evski N, Kljajic´ P (1997a) Insecticide susceptibility of the Colorado potato beetle in the vicinity of Belgrade, Yugoslavia. Acta Hortic 462:983–990
15 Peric´ I, Milosˇ evski N, Kljajic´ P, Sˇestovic´ M (1997b) Evolucija rezistentnosti cˇetiri populacije krompirove zlatice prema karbarilu i fosmetu. Pesticidi 12:269–276 Raymond M. (1985): Presentation d’un programme d’analyse logprobit pour micro- ordinnateur. Cah ORSTOM, Ser Entomol Med Parasitol 22:117–121 Roush RT, Hoy CW, Ferro DN, Tigney WM (1990) Insecticide resistance in the Colorado potato beetle (Coleoptera: Chrysomelidae): influence of crop rotation and insecticidal use. J Econ Entomol 83:315–319 Sˇestovic´ M (1972) Rezistentnost krompirove zlatice, Leptinotarsa decemlineata (Say), prema insekticidima. Doktorska disertacija, Poljoprivredni fakultet, Belgrade-Zemun Sˇestovic´ M, Peric´ I (1977) Spektar rezistentnosti krompirove zlatice, Leptonotarsa decemlineata (Say), prema insekticidima. VIII Savetovanje o primeni pesticida u zasˇ titi bilja, higijeni i u veterini-Porecˇ. Zbornik Radova 2:481–495 Stankovic´ S (2002) Monitoring methods and resistance levels of Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) in Serbia. Master thesis, University of Belgrade, Serbia, pp 1–64 Stewart J, Kennedy G, Sturz A (1997) Incidence of insecticide resistance in populations of Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), on Prince Edward Island. Can Entomol 129:21–26 Turnbull SA, Tolman JH, Harris CR (1988) Colorado potato beetle resistance to insecticides in Ontario, Canada. Brighton Crop Protection Conference—Pests and Diseases 1:457–463 Zabel A (1991) Aktivnost esteraza i otpornost prema organofosfornim insekticidima u krompirove zlatice [Leptinotarsa decemlineata (Say)]. Doktorska disertacija, Poljoprivredni fakultet, Novi Sad
Zabel A, Kostic´ M (1988) Ispitivanje efikasnosti i perzistentnosti nekih novih insekticida u suzbijanju krompirove zlatice [Leptinotarsa decemlineata (Say)]. Zasˇ t Bilja 184:171–182 Zabel A, Kostic´ M, Manojlovic´ B (1997a) Efikasnost insekticida iz grupe inhibitora rasta insekata u suzbijanju populacije krompirove zlatice rezistentne na organofosforne i karbamatne insekticide. Zasˇ t Bilja 219:107–117 Zabel A, Kostic´ M, Sivcˇev I, Draganic´ M, Indjic´ D (1997b) Susceptibility of the Colorado potato beetle [Leptinotarsa decemlineata (Say), (Coleoptera: Chrysomelidae)] to applied insecticides in the Republic of Serbia. Acta Hortic 462:397–405 Zabel A, Zˇivanovic´ M, Sˇestovic´ M, Kostic´ M (1998) ALiE activity of larvae Leptinotarsa decemlineata (Say) to dithiophosphates. 9th International Congress on Pesticide Chemistry, vol. 1:4B– 028, London, UK (abstracts) Zabel A, Rajkovic S, Manojlovic B, Stankovic S, Veljkovic I (2000a) New pesticides in potato protection against the Colorado potato beetle [Leptinotarsa decemlineata (Say)] and late blight [Phytophtora infestans (Mont. de Bary)] on Potato. 2nd Balkan Symposium on Vegetables and Potatoes. Thessaloniki, Greece (abstracts) Zabel A, Rajkovic S, Manojlovic B, Stankovic S, Kostic M (2000b) Nenicotinoids in the control of Leptinotarsa decemlineata (Say). 2nd Balkan Symposium on Vegetables and Potatoes. Thessaloniki, Greece (abstracts) Zehnder G W, Evanylo GK (1989) Influence of extent and timing of Colorado potato beetle (Coleoptera: Chrysomelidae) defoliation on potato tuber production in eastern Virginia. J Econ Entomol 82:948–953 Zhao JZ, Bishop BA, Grafius EJ (2000) Inheritance and synergism of resistance to imidacloprid in the Colorado potato beetle (Coleoptera: Chrysomelidae). J Econ Entomol 93:1508–1514